PROJECT SUMMARY Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are neutrophilic inflammatory lung diseases with unacceptably high rates of morbidity and mortality. Our supportive data indicate that genetic ablation and pharmacologic inhibition of NFATc3 is beneficial in terms of tighter pulmonary barrier function, decreased cytokine release and neutrophilic inflammation, improved arterial oxygenation, and better survival in mouse models of ARDS/ALI. Based on these and other clinically relevant supportive data, we posit that NFATc3 activation in macrophages induces inflammatory gene expression that mediates the initiation, intensity and duration of ALI/ARDS. We propose three specific aims to address this hypothesis: Specific Aim 1: To identify the mechanism(s) by which NFATc3 deletion protects against ALI/ARDS. We will employ genetically defined mice that globally lack NFATc3 subjected to a well-defined murine model of ALI/ARDS. We will also utilize established methods of NFATc3 KO/WT bone marrow chimera mice, macrophage passive adoptive transfer of lung macrophages, and we will construct novel conditional macrophage selective NFATc3-/- mice to identify the impact of macrophage NFATc3 deficiency on the generation of pulmonary edema, arterial oxygen saturation, and mortality. Specific Aim 2: To determine the mechanisms by which macrophage NFATc3 activation contributes to the development of permeability pulmonary edema. In order to determine how macrophages contribute to the pulmonary edema and severe hypoxemia in ALI/ARDS, we propose to examine the role of NFATc3 regulation of macrophage expression of TNF-related apoptosis-inducing ligand (TRAIL), which inhibits alveolar fluid clearance (AFC), in addition to lung macrophage production of mediators that disrupt barrier function of pulmonary microvascular endothelium. Specific Aim 3: To determine the efficacy of a novel cell permeable peptide NFAT inhibitor in protecting against ALI/ARDS-like pathophysiology in mouse models. Our group has developed a novel NFAT inhibitor, CP9-ZIZIT, that has an impressively low Kd value of 2.6 nM, better cell permeability, higher binding affinity, and improved stability against proteolysis than other available agents. We expect to show that treatment with CP9-ZIZIT alone or in combination with a PARP1 inhibitor will attenuate pulmonary inflammation and alveolar capillary barrier disruption. We expect that this research will provide valuable preclinical data that blocking NFATc3 activation in macrophages is an effective therapeutic agent for preventing ARDS in a high-risk patient population and improving the clinical outcome of patients suffering from moderate and severe ARDS/ALI.